| Objectives:To investigate the effect of scutellarin on the polarization of activated microglia and the regulatory effect of MAPKs signaling pathway on the polarization of M2 microglia,and to further clarify the mechanism of scutellarin promoting the neurotrophic and protective responses mediated by activated M2 microglia.Methods:In vitro,BV-2 microglia cell line was conducted,and LPS was used to activate BV-2 microglia.The cells were divided into three groups:control group,LPS group and LPS+scutellarin(0.54mM,Monomer,the main active component of breviscapine)group.The expression changes of MAPKs proteins(phosphorylated and non-phosphorylated:p-p38,P38,p-JNK,JNK,p-ERK1/2 and ERK1/2)and M2 microglia markers(CD206,Argl,YM1/2,IL-4 and IL-10)were detected by immunofluorescence staining and Western Blot.After pretreatment with MAPKs signaling pathway inhibitors,the expression of M2 microglia marker Argl was detected in control group,LPS group and LPS+scutellarin group.In vivo,the middle cerebral artery occlusion(MCAO)model was replicated by minimally invasive craniotomy.The rats were randomly divided into Sham group,MCAO group,and MCAO+scutellarin group(100mg/kg,The main active component of scutellarin is scutellarin,which accounts for more than 90%),with a total of 3 groups(n=5 for each group).The expression of M2 microglia markers and MAPKs signals pathway in the cerebral cortex of rats in each group were detected by immunofluorescence staining.The expression of MAPKs signal and M2 microglia markers in the cortex of rats was detected by Western Blot.Results:(1)In vitro:1.Western Blot showed that the expression of CD206,YM1/2,Arg1 and IL-10 were significantly increased in the LPS group,with significant differences compared with control group(p<0.01);The expression of CD206,YM1/2,Argl,IL-10 and IL-4 were obviously increased after pretreat with scutellarin.There were significant differences between the LPS group and the LPS group(p<0.01).(2)Immunofluorescence staining showed that the expression of CD206,YM1/2,IL-10,IL-4 and Argl were induced in the LPS group,which was statistically significant compared,with the control group(p<0.05);The expression of CD206,YM1/2,IL-10,IL-4 and Argl in BV-2 microglia were significantly increased after scutellarin pretreatment,with statistically significant differences compared with the LPS group(p<0.05).(3)The results of Immunofluorescence staining showed that Arg1,p-JNK and p-p38 were enhanced in LPS group,which was statistically significant compared with the Con group(p<0.05);Argl expression was significantly increased and the expression of p-JNK and p-p38 was significantly decreased after scutellarin pretreatment,which was statistically different from that of the LPS group(p<0.05).(4)The Immunofluorescence staining showed that the expression of Argl and p-ERK1/2 were enhanced in the LPS group,which was statistically significant compared with the Con group(p<0.05);After pretreatment with scutellarin,Argl was significantly increased and p-ERK 1/2 was also significantly increased,which was statistically different from that of the LPS group(p<0.05).(5)Western Blot showed that the expression of YM1/2;Argl and IL-10 were significantly increased in microglia pretreated with scutellarin in LPS activated microglia,which were significantly different from those in the LPS group(p<0.01);After pretreatment with p38 inhibitor(SB203580)and JNK inhibitor(HY-12041),YM1/2,Arg1 and IL-10 were significantly increased,which was statistically significant compared with the LPS+scutellarin group(p<0.01).(6)Western Blot showed that the expression of YM1/2,Argl and IL-10 were significantly increased in microglia pretreated with scutellarin in LPS activated microglia,which were significantly different from LPS group(p<0.01);After pretreatment with ERK1/2 inhibitor(HY-112287),YM1/2,Argl and IL-10 were significantly decreased compared with LPS+S group(p<0.01).(2)In vivo,Western Blot showed that:in 1d MCAO rat models,the expression of YM1/2,Argl and IL-10 in the MCAO group was significantly increased,and there were significant differences between Sham group(p<0.01),there was no significant difference in the expression of CD206 and 1L-4 between the sham group and the sham group.The expression of YM1/2,Argl,and IL-4 increased after scutellarin treatment,and was obviously different from that of MCAO group(p<0.05),the protein expression of CD206 and IL-10 was not significantly different from that of MCAO group.In the 7d MCAO rat models,CD206,YM1/2,Argl and IL-10 were significantly increased in MCAO group,with significant differences compared with Sham group(p<0.01),CD206,YM1/2,Argl and IL-4 were significantly increased after treatment with scutellarin and there were significant difference between the MCAO group and the MCAO group(p<0.05),there was no significant difference in IL-10 protein expression between MCAO group and MCAO+S group.In the 3d MCAO rat models,the expression of CD206,YM1/2,Argl and IL-10 in MCAO group were significantly increased,and the differences between MCAO group and Sham group were significant(p<0.01);The expression of CD206,YM1/2,Argl,IL-10 and IL-4 were significantly increased after treatment with scutellarin.There were significant differences between MCAO group and MCAO+S group(p<0.01).Conclusions:(1)CD206,Argl,YM1/2,IL-4,and IL-10 were highly expressed in BV-2 microglia activated by LPS and activated microglia after cerebral ischemia in rats.(2)The expression of M2 microglial markers CD206,Arg1,YM1/2,IL-4,and IL-10 was significantly increased by scutellarin in LPS-activated microglia.(3)Scutellarin may regulate M1/M2 polarization of microglia through MAPKs signaling pathway. |
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